Objective and Aims for Project 3 The internalization and recycling of cell surface receptors, glucose transporters, and other plasma membrane proteins is regulated by phosphoinositide metabolism as well as GTPases of the Rab and Arf families. Defects in these regulatory mechanisms have been implicated in a variety of disease states including cancer and type II diabetes. The long term objective of Project 3 is to characterize poorly understood mechanisms that integrate phosphoinositide signaling and Rab/Arf GTPase activation with the dynamic molecular assemblies that mediate/regulate trafficking events at the plasma membrane and within the endosomal system. To achieve this objective, we will combine crystallographic, biochemical, biophysical, and mutational studies on purified complexes with complementary structure-function analyses in collaboration with Projects 1, 2, 4, and the imaging core. Building on the observations of the previous funding period and extensive new preliminary data, we will: (Aim 1) examine the structural determinants of autoregulation and phosphoinositide dependent membrane targeting in the Grp1 family of Arf GTPase exchange factors; (Aim 2) investigate the mechanism of autoregulation, membrane targeting, and Rab activation by the Rabex-5/Rabaptin-5 complex and Rin1;and (Aim 3) explore the structural bases for regulation of endocytic recycling by phosphoinositide dependent and independent complexes of EHD proteins with Rabenosyn-5, EHBP1, and FIP2. Relevance to Public Health Both type II diabetes and cancer involve defects in the mechanisms by which cell surface receptors for insulin and growth factors communicate with the molecular machinery that controls cell metabolism and growth. By studying these mechanisms comprehensively at the structural, molecular, and cellular levels through collaborative projects the combine the expertise of structural, molecular, and cell biologists we hope to identify novel mechanism-based strategies that can be exploited for therapeutic intervention.